Sterile intra-amniotic inflammation is commonly observed in patients with spontaneous preterm labor, a syndrome that commonly precedes preterm birth, the leading cause of perinatal morbidity and mortality worldwide. However, the mechanisms leading to sterile intra-amniotic inflammation are poorly understood and no treatment exists for this clinical condition. Herein, we investigated whether the alarmin S100B could induce sterile intra-amniotic inflammation by activating the NLRP3 inflammasome, and whether the inhibition of this pathway could prevent preterm labor/birth and adverse neonatal outcomes. We found that the ultrasound-guided intra-amniotic administration of S100B induced a 50% rate of preterm labor/birth and a high rate of neonatal mortality (59.7%) without altering the fetal and placental weights. Using a multiplex cytokine array and immunoblotting, we reported that S100B caused a proinflammatory response in the amniotic cavity and induced the activation of the NLRP3 inflammasome in the fetal membranes, indicated by the upregulation of the NLRP3 protein and increased release of active caspase-1 and mature IL-1β. Inhibition of the NLRP3 inflammasome via the specific inhibitor MCC950 prevented preterm labor/birth by 35.7% and reduced neonatal mortality by 26.7%. Yet, inhibition of the NLRP3 inflammasome at term did not drastically obstruct the physiological process of parturition. In conclusion, the data presented herein indicate that the alarmin S100B can induce sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes by activating the NLRP3 inflammasome, which can be prevented by inhibiting such a pathway. These findings provide evidence that sterile intra-amniotic inflammation could be treated by targeting the NLRP3 inflammasome.
Intra-amniotic inflammation is strongly associated with spontaneous preterm labor and birth, the leading cause of perinatal mortality and morbidity worldwide. Previous studies have suggested a role for the NLRP3 (NLR family pyrin domain-containing protein 3) inflammasome in the mechanisms that lead to preterm labor and birth. However, a causal link between the NLRP3 inflammasome and preterm labor/birth induced by intra-amniotic inflammation has not been established. Herein, using an animal model of lipopolysaccharide-induced intra-amniotic inflammation (IAI), we demonstrated that there was priming of the NLRP3 inflammasome (1) at the transcriptional level, indicated by enhanced mRNA expression of inflammasome-related genes (Nlrp3, Casp1, Il1b); and (2) at the protein level, indicated by greater protein concentrations of NLRP3, in both the fetal membranes and decidua basalis prior to preterm birth. Additionally, we showed that there was canonical activation of the NLRP3 inflammasome in the fetal membranes, but not in the decidua basalis, prior to IAI-induced preterm birth as evidenced by increased protein levels of active caspase-1. Protein concentrations of released IL1β were also increased in both the fetal membranes and decidua basalis, as well as in the amniotic fluid, prior to IAI-induced preterm birth. Finally, using the specific NLRP3 inhibitor, MCC950, we showed that in vivo inhibition of the NLRP3 inflammasome reduced IAI-induced preterm birth and neonatal mortality. Collectively, these results provide a causal link between NLRP3 inflammasome activation and spontaneous preterm labor and birth in the context of intra-amniotic inflammation. We also showed that, by targeting the NLRP3 inflammasome, adverse pregnancy and neonatal outcomes can be significantly reduced.
Abstract During mitosis, cells undergo symmetrical cell division, while oocyte meiotic maturation undergoes two consecutive, asymmetric divisions that generate a totipotent haploid oocyte and two small polar bodies not involved in DNA replication. This specialized division allows most maternal components to be maintained in the oocytes for early embryo development. Nuclear positioning, germinal vesicle breakdown, spindle migration, spindle rotation, chromosome segregation, and polar body extrusion are the most critical cellular processes during oocyte meiosis I and II, and a growing number of studies primarily using the mouse oocyte model revealed that actin filaments were critical for these processes, especially for spindle migration. Several important molecules have been reported to be involved in these processes. One family of molecules are the small GTPases, such as Rho GTPases, Ran GTPases, and Rab GTPases and another are the actin nucleators, such as the formin family and the Arp2/3 complex. The present review summarizes recent progress made regarding the roles of actin filaments in the asymmetric oocyte division. Actin filaments widely involve into multiple cellular processes such as nuclear positioning, germinal vesicle breakdown, spindle migration, chromosome segregation, spindle rotation and polar body extrusion in oocyte mammalian meiosis.
Abstract The objective of this work was to determine the role of mitochondria in the loss of oocyte quality with maternal aging. Our results show that mitochondrial DNA (mtDNA) copy number and function are reduced in eggs from aged mice after both in vivo and in vitro maturation. Higher incidences of spindle abnormalities were observed in old eggs. However, no correlation with egg ATP content was found. In vitro matured eggs from aged mice did not have a normal cortical distribution of active mitochondria and were subject to increased oxidative stress due to higher levels of reactive oxygen species and lower expression of glutamate-cysteine ligase, catalytic subunit (Gclc). Supplementation of antioxidants during in vitro maturation of old eggs mitigated this affect, resulting in increased mtDNA copy number and mitochondrial function, a mitochondria distribution pattern similar to young eggs, and improved chromosomal alignment. Eggs from women of advanced maternal age (AMA) had lower mitochondrial function than eggs from young women, although both age groups displayed a cortical distribution pattern of active mitochondria. In contrast to the mouse, human eggs from AMA women had higher mtDNA copy number than eggs from young women following in vitro maturation. In summary, oocytes of older females are more susceptible to perturbations in mitochondrial number and function, which are associated with increased spindle abnormalities and oxidative stress during in vitro maturation. These results demonstrate that oocyte mitochondria play a critical role in age-related infertility. A decrease in mitochondrial copy number and function affects oocyte quality in aged females, and is related to increased spindle abnormalities and oxidative stress.
The seminal vesicles (SV) can be infected by microorganisms, thereby resulting in vesiculitis and impairment in male fertility. Innate immune responses in SV cells to microbial infections, which facilitate vesiculitis, have yet to be investigated. The present study aims to elucidate pattern recognition receptor (PRR)-mediated innate immune responses in SV epithelial cells. Various PRRs, including Toll-like receptor 3 (TLR3), TLR4, cytosolic RNA and DNA sensors, are abundantly expressed in SV epithelial cells. These PRRs can recognize their respective ligands, thus activating nuclear factor kappa B and interferon regulatory factor 3. The PRR signaling induces expression of pro-inflammatory cytokines, such as Tnfa and Il6, chemokines Mcp1 and Cxcl10, and type 1 interferons Ifna and Ifnb. Moreover, PRR-mediated innate immune responses upregulated the expression of microsomal prostaglandin E synthase and cyclooxygenase 2, but they downregulated semenogelin-1 expression. These results provide novel insights into the mechanism underlying vesiculitis and its impact on the functions of the SV.
The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation, the wound is repaired, however this process is poorly understood. In epithelial tissues, wound repair is mediated by an epithelial-to-mesenchymal transition (EMT). Transforming Growth Factor Beta-1 (TGFβ1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We therefore hypothesized that TGFβ1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFβ1 induced an EMT mediated by TGFβRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFβ1 and, when overexpressed, was shown to increase OSE cell migration. A PCR array of TGFβ signaling targets determined Cox2 to be most highly induced by TGFβ1. Constitutive Cox2 expression modestly increased migration and robustly enhanced cell survival, under stress conditions similar to those observed during wound repair. The increase in Snail and Cox2 expression with TGFβ1was reproduced in human OSE cultures, suggesting these responses are conserved between mouse and human. Finally, the induction of Cox2 expression in OSE cells during ovulatory wound repair was shown in vivo, suggesting TGFβ1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFβ1 promotes ovulatory wound repair by induction of an EMT and activation of a COX2-mediated pro-survival pathway. Understanding ovulatory wound repair may give insight into why ovulation is the primary non-hereditary risk factor for ovarian cancer.
Spalt-like 1 (SALL1) and spalt-like 3 (SALL3) are multi-zinc finger transcription factors that play an essential role in regulating developmental processes and organogenesis in many species. However, the functional role of SALL1 (csal1) and SALL3 (csal3) in chicken prehierarchical follicle (PF) development is unknown. This study aimed to explore the potential role and mechanism of csal1 and csal3 in granulosa cells (GCs) proliferation, differentiation, and follicle selection within the PFs of hen ovary. Our data demonstrated that the csal1 and csal3 transcriptions were highly expressed in GCs of the various-sized PFs and their proteins were mainly localized in the cytoplasm of granulosa cells and oocytes of the examined PFs as well as in the ovarian stroma and epithelium. It initially revealed that both csal1 and csal3 may be involved in chicken PF development via a translocation mechanism. Furthermore, our results showed an abundance of CCND1, Bcat, StAR, CYP11A1, and FSHR mRNA in GCs and the proliferation levels of GCs from the PFs were significantly increased by siRNA-mediated knockdown of csal1 or/and csal3. Conversely, the overexpression of csal1 or/and csal3 in the GCs led to a remarkably decreased mRNA expression of the five genes correlating with an decline in cell proliferation levels. Moreover, csal1 and csal3 togather exert a much stronger effect on the regulation than any of csal1 or csal3. These results indicated that csal1 and csal3 play synergistic inhibitory roles on GC proliferation, differentiation, and steroidogenesis during PF development in vitro. The current data provides a basis for further investigation of molecular mechanisms of csal1 and csal3 in controlling the PF development and growth of hen ovary in vivo.
The oviduct plays a crucial role in fertilization and early embryo development providing the microenvironment for oocyte, spermatozoa, and early embryo. Since dairy cow fertility declined steadily over the last decades, reasons for early embryonic loss have gained increasing interest. Analyzing two animal models, this study aimed to investigate the impact of genetic predisposition for fertility as well as of metabolic stress on the protein composition of oviduct fluid (OF). A metabolic model comprised maiden Holstein heifers (MH) and postpartum lactating (Lact) and non-lactating (Dry) cows, while a genetic model consisted of heifers from the Montbéliarde (MBD) breed and Holstein heifers with low (LFH) and high (HFH) fertility index. In a holistic proteomic analysis of OF from all groups using nano-LC-MS/MS analysis and label-free quantification, we were able to identify 1976 proteins, amongst which 143 showed abundance alterations in the pairwise comparisons within both models. Most differentially abundant proteins were revealed between LFH and MBD (52) in the genetic model and between Lact and MH (19) in the metabolic model, demonstrating a substantial effect of genetic predisposition for fertility as well as metabolic stress on the OF proteome. Functional classification of affected proteins revealed actin binding, translation and immune system processes as prominent GO clusters. Notably, Actin-related protein 2/3 complex subunit 1B (ARPC1B) and the three immune system-related proteins SERPIND1, IGK protein and Alpha-1-acid glycoprotein (AGP) were affected in both models, suggesting that abundance changes of immune-related proteins in OF play an important role for early embryonic loss.
Mammalian oocytes and eggs are transcriptionally quiescent and depend on post-transcriptional mechanisms for proper maturation. Post-transcriptional mRNA modifications comprise an important regulatory mechanism that can alter protein and miRNA recognition sites, splicing, stability, secondary structure, and protein coding. We discovered that fully-grown mouse germinal vesicle (GV) oocytes and metaphase II (MII) eggs display abundant inosine mRNA modifications compared to growing oocytes from postnatal day 12 (PND12) oocytes. These inosines were enriched in mRNA protein coding regions (CDS) and specifically located at the third codon base or wobble position. Inosines, observed at lower frequencies in CDS of somatic tissues, were similarly enriched at the codon wobble position. In oocytes and eggs, inosines modifications lead primarily to synonymous changes in mRNA transcripts. Inosines may ultimately affect maternal mRNA stability by changing codon usage, thereby altering translation efficiency and translationally coupled mRNA degradation. These important observations advance our understanding of post-transcriptional mechanisms contributing to mammalian oocyte maturation.
Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB) layer. Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role in STB formation through its fusogenic properties, but also possesses an active immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also have an immunosuppressive effect, placental exosomes were incubated with activated Jurkat T cells and PBMCs. Upon quantification of Th1 cytokines in the supernatants, T cell activation was severely reduced. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCS were less suppressive when compared to exosome derived from VCT transfected with control siRNA. Our results suggest that Syn-2 could be an important immune regulator both locally and at the systemic level, via its association with placental exosomes.